Extracellular matrix metalloproteinase inducer (EMMPRIN, also known as CD147, basigin, neurothelin, OK blood group antigen, Leukocyte activation antigen M6, and Tumor cell-derived collagenase stimulatory factor TCS), is a tumor-promoting protein expressed by both tumor and stromal cells that has been shown to induce both the pro-angiogenic vascular endothelial growth factor (VEGF) and the extracellular matrix (ECM) degrading matrix metalloproteinases (MMPs), including MMP-9. In addition, EMMPRIN is involved in tumor metabolism via its interactions with the lactate transporters MCT-1 and MCT-4, in tumor drug resistance via its interactions with cyclophilins and the MDR1 protein (P-gp), and in leukocyte chemotaxis via its interactions with the extracellular cyclophilin A. Clinical trials that used a wide range of MMPs inhibitors failed to show the inhibition of tumor progression, probably due to the redundancy of MMPs in tumor tissues, and anti-VEGF antibodies provide only temporary inhibition of tumor progression. EMMPRIN, with its multiple functions in tumor survival and growth, may serve as an attractive target in cancer immunotherapy, so that immunizing against this one molecule may simultaneously affect many of its functions in promoting disease.
Both tumor cells and the infiltrating leukocytes, particularly macrophages, secrete high levels of tumor-promoting factors, such as the pro-angiogenic vascular endothelial cell growth factor (VEGF) and the ECM-degrading MMPs, both crucial for tumor progression, invasiveness, metastasis and angiogenesis. High levels of MMPs, particularly MMP-9, release and activate VEGF that is trapped by the ECM, and allow migration of leukocytes, metastatic tumor cells and endothelial cells. VEGF is a chemoattractant for macrophages, and a regulator of MMP-9. Thus, a positive feedback loop exists whereby MMP-9 and VEGF enhance each other (Hollborn et al., Invest Ophthalmol Vis Sci. 2007; 48:4360-4367).
EMMPRIN has a critical role in spermatogenesis, female fertilization, and retinal development (Weidle et al., 2010; 7:157-169). In the tumor context, EMMPRIN is expressed by both tumor and stromal cells, including macrophages (Nabeshima et al., Pathol Int. 2006; 56:359-367., Yan et al., Thromb Haemost. 2005; 93:199-204), and functions to enhance the expression of VEGF and MMPs, including MMP-9, in solid tumors. Over-expression of EMMPRIN increases invasiveness of tumor cells, and neutralizing antibody against EMMPRIN reduces the level of cellular MMP-9 and VEGF expression. Additional functions of EMMPRIN are also important for tumor survival and progression, such as its ability to chaperone the monocarboxylate transporters MCT-1 and MCT-4 that transport lactate out of the cells, its involvement in the multidrug resistance (MDR) phenotype through its interactions with cyclophilin A and P-gp (Kanekura T, Chen X. J Dermatol Sci. 2010; 57:149-154), and its ability to recruit leukocytes into the tumor through its interactions with cyclophilin A (Yurchenko V et al., Clin Exp Immunol. 2010; 160:305-317). Thus, targeting this single molecule may have many beneficial effects in cancer immunotherapy.
The regulation of EMMPRIN expression is generally unknown, although EGFR and angiotensin II were implicated in EMMPRIN expression in fibroblasts and macrophages, respectively. Equally unknown is the signaling pathway(s) that EMMPRIN initiates to induce MMP-9 and VEGF, although homophilic EMMPRIN:EMMPRIN binding, surface-anchored or soluble (Belton et al., J Biol. Chem. 2008; 283:17805-17814), was shown to elevate them and promote tumor growth and angiogenesis (Tang et al. Cancer Res. 2005; 65:3193-3199). The ability to elevate MMP-9 was mapped to the first of two heavily glycosylated extracellular domains (EC-I, EC-II or D-I and D-II, Belton R J, at el, ibid), but the precise epitopes are not known, nor is the region that regulates VEGF induction. In addition, EMMPRIN has a longer isoform that has an additional membrane-distal Ig-like domain (EC-0 or D-0, Muramatsu T, Expert Opin. Ther. Targets, 2012, 16(10):999-1011), which is expressed mostly in the retina, and is presumed to function in the complex transporting lactate.
Several attempts have been made to target EMMPRIN. Knockdown of EMMPRIN by small interfering RNA (siRNA) decreased MMP-9 and diminished invasiveness of a prostate cancer cell line in vitro (Wang et al. Cancer Biol Ther. 2006; 5:608-614). Monoclonal antibodies (mAbs) against the extracellular fragment of EMMPRIN reduced or elevated MMP-2 in human fibroblasts co-cultured with hepatocellular carcinoma cells (HCC) in vitro, suggesting that different EMMPRIN epitopes may be involved in MMPs induction. Another anti-EMMPRIN mAb reduced MMPs and VEGF secretion in co-culture of head and neck cancer cell line with fibroblasts (Dean et al. Clin Cancer Res. 2009; 15:4058-4065). Licartin, the F(ab)2 fragment of an anti-EMMPRIN antibody, delayed recurrence of hepatoma after transplantation in human patients (Xu et al. Hepatology. 2007; 45:269-276), suggesting the possibility of adding passive immunization with anti-EMMPRIN to the already existing immunotherapy approaches (e.g. anti-Her/neu in breast cancer). However, anti-EMMPRIN antibodies were raised against the entire extracellular fragment, so that the epitopes responsible for the different activities of EMMPRIN remain unidentified. This may be important when designing anti-EMMPRIN immunotherapy, as targeting different epitopes may have different effects on physiological or on tumor activities of the protein. Experimental active immunization protocols exist, such as administration of whole tumor lysates, tumor antigen peptides, antigen-pulsed dendritic cells or naked-DNA plasmids, but active immunization to EMMPRIN has not yet been reported.
Angiogenesis
Angiogenesis is an important cellular event in which vascular endothelial cells proliferate, prune and reorganize to form new vessels from preexisting vascular networks. There is compelling evidence that the development of a vascular supply is essential for normal and pathological proliferative processes and inflammation. The vascular compartment is necessary not only for organ development and differentiation during embryogenesis, but also for wound healing, tissue repair and reproductive functions in the adult.
Angiogenesis is also implicated in the pathogenesis of a variety of disorders, including but not limited to, tumors, proliferative retinopathies, age-related macular degeneration, rheumatoid arthritis, and psoriasis. Angiogenesis is essential for the growth of most primary tumors and their subsequent metastasis. Tumors can absorb sufficient nutrients and oxygen by simple diffusion up to a size of 1-2 mm, at which point their further growth requires the elaboration of a vascular supply. This process is thought to involve recruitment of the neighboring host mature vasculature to begin sprouting new blood vessel capillaries, which grow toward, and subsequently infiltrate, the tumor mass. In addition, tumor angiogenesis involves the recruitment of circulating endothelial precursor cells from the bone marrow to promote neovascularization. It is suggested that the angiogenesis process is regulated by a balance between pro- and anti-angiogenic molecules, and is derailed in various diseases, especially cancer.
Angiogenesis (which involves secretion of pro-angiogenic factors such as VEGF) is initiated in many inflammatory diseases, especially in chronic inflammatory diseases.
VEGF, the most prevalent form of which is VEGF-A or vascular permeability factor (VPF), has been reported as a pivotal regulator of both normal and abnormal angiogenesis. Human VEGF is a 32-42 kilodalton (kDa) dimeric glycoprotein that mediates vasodilatation, increased vascular permeability and endothelial cell proliferation.
VEGF mRNA is overexpressed by the majority of human tumors examined. Given its central role in promoting tumor growth, VEGF is a very attractive target for therapeutic intervention. Indeed, a variety of therapeutic strategies aimed at blocking VEGF or its receptor signaling system are being developed for the treatment of neoplastic diseases.
MMPs are a family of highly homologous protein-degrading zinc-dependent endopeptidases. This family includes more than 25 members that can be divided into collagenases (MMP-1, -8, and -13), gelatinases (MMP-2 and -9), stromelysins (MMP-3 and -10), matrilysins (MMP-7 and -26), and membrane-type MMPs (MMP-14 to −17 and -24). MMPs are important in many normal biological processes including embryonic development, ovulation and implantation, bone growth, angiogenesis, inflammation, and wound healing, as well as in pathological processes such as cancer, autoimmune diseases, atherosclerosis and ischemic diseases, Alzheimer's disease and tissue destruction. MMPs collectively cleave most, if not all, of the constituents of the ECM and are involved in the breakdown and remodeling of many tissues and organ.
All inflammatory diseases require cell movement through the ECM. In particular, leukocytes must migrate from the blood vessels through the basement membrane (BM) and the ECM in order to reach the inflammatory site. This requires degradation or remodeling of the ECM and BM, which employs MMPs, including MMP-9. Additionally, many inflammatory diseases require new or additional blood supply to the inflammatory site, because the increased number of cells in the leukocyte infiltrate has higher metabolic demands that demand more oxygen and nutrients.
EMMPRIN and Anti-EMMPRIN Strategies:
Ku et al (Scandinavian Journal of Immunology 2007, 65, 435-443) describes epitope mapping of several mAbs against EMMPRIN and suggest that the fragment having the sequence AAGTVFTTVEDLGSKILLTCSLNDSATEV plays a critical role in the functions of this protein on MMP secretion and tumor invasion.
Kawakami et al. (MBC cancer 2011, 11:300, http://www.biomedcentral.com/1471-2407/11/300) demonstrated, using a polypeptide of 61 amino acids corresponding to the EC-I domain of EMMPRIN that this domain can mimic EMMPRIN activity in term of stimulating MMP-2 production by fibroblasts, when substituted with chitobiose, the disaccharide with which N-glycosylation starts.
U.S. Pat. No. 7,452,680 discloses a fusion protein between the entire sequence of rodent CD147 and human MCT1 transporter.
U.S. Pat. No. 7,005,500 discloses a polymorphic variant of human basigin which contains two immunoglobulin domains, one of which is a domain of 56 amino acids. The new variant, named BASI2, displays similar biological activities as basigin, but displays enhanced kinetic parameters during protein-protein interactions.
None of the publications disclose the specific antigenic determinant of EMMPRIN disclosed in the present invention, peptides therefrom, and specific antibodies that bind to said antigenic determinant. There is an unmet need to provide isolated immunogenic and immunotherapeutic peptides and antibodies directed against them that can be used to diagnose, prevent and treat medical conditions such as proliferative and inflammatory diseases associated with expression of the protein EMMPRIN and with regulation of VEGF and MMP levels.